This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Autologous chondrocyte transplantation (ACT) has been shown, in long-term follow-up
studies, to be a promising treatment for the repair of isolated cartilage lesions.
The method is based on an implantation of in vitro expanded chondrocytes originating from a small cartilage biopsy harvested from a non-weight-bearing
area within the joint. In patients with osteoarthritis (OA), there is a need for the
resurfacing of large areas, which could potentially be made by using a scaffold in
combination with culture-expanded cells. As a first step towards a cell-based therapy
for OA, we therefore investigated the expansion and redifferentiation potential in vitro of chondrocytes isolated from patients undergoing total knee replacement. The results
demonstrate that OA chondrocytes have a good proliferation potential and are able
to redifferentiate in a three-dimensional pellet model. During the redifferentiation,
the OA cells expressed increasing amounts of DNA and proteoglycans, and at day 14
the cells from all donors contained type II collagen-rich matrix. The accumulation
of proteoglycans was in comparable amounts to those from ACT donors, whereas total
collagen was significantly lower in all of the redifferentiated OA chondrocytes. When
the OA chondrocytes were loaded into a scaffold based on hyaluronic acid, they bound
to the scaffold and produced cartilage-specific matrix proteins. Thus, autologous
chondrocytes are a potential source for the biological treatment of OA patients but
the limited collagen synthesis of the OA chondrocytes needs to be further explained.

Introduction

Adult articular cartilage consists of a delicate system of cells and matrix proteins,
which have the function of creating a viscoelastic tissue with high biomechanical
stability and low friction. Even though the cartilage is exposed to continuous mechanical
wear, there is surprisingly low turnover in cells and extracellular matrix [1], which could be a reason for the inability of adult articular cartilage to respond
to injuries and subsequently repair lesions. This low potential of self-repair has
led to the development of several techniques such as mosaic plastic, microfracture,
periosteal transplantation and autologous chondrocyte transplantation (ACT), all seeking
to create a functional and painless repair of articular cartilage defects.

In ACT, culture-expanded chondrocytes are transplanted under a cover of periosteum
[2]; the method was initially aimed at the treatment of small isolated lesions. However,
10 years later, the indication has been expanded to include lesions up to 20 cm2 in size. This first generation of cell-based treatment has been followed by a second
or third generation, consisting of culture-expanded cells loaded on a membrane or
into a biodegradable scaffold before implantation [3,4]. One major advantage in using scaffolds as cell carriers is that the cells can be
positioned in the lesion, thereby ensuring that the cells become evenly distributed
in the defect. Subsequently, the degradation time of the scaffold needs to be controlled.
This can be made by different combinations of poly-L-lactic acid and poly-(lactic-co-glycollic
acid) [5] or by the esterification of hyaluronic acid [6,7]. The scaffold made of hyaluronic acid has additionally been shown to degrade into
chondrogenically active components [8].

Another major advantage of using a scaffold for delivery of the cells is the potential
for treating larger defects. This is especially interesting for young (under 60 years
old) and active patients with developed osteoarthritis (OA), who at present lack an
appropriate treatment alternative. The aetiology of OA has been suggested to contain
a phenotypic alteration of the chondrocytes [9] and disturbance in the proteoglycan metabolism due to systematic, mechanical or unknown
reasons. Chondrocytes isolated from OA cartilage have been shown to be more metabolically
active than cells isolated from non-OA regions in the same joint [10], whereas chondrocytes isolated from less severe grades of OA cartilage synthesize
normal matrix components [11].

When chondrocytes are isolated from their three-dimensional (3D) environment in the
articular cartilage and expanded in monolayer cultures, the cells dedifferentiate
and gradually lose their specific phenotype [12,13]. We have shown previously that dedifferentiated cells from ACT patients have the
ability to differentiate into several mesenchymal phenotypes [14] and that during redifferentiation towards the chondrogenic phenotype the cells express
genes known to be involved in the embryonic formation of cartilage [15].

We therefore proposed, as a first step towards cell-based treatments for OA, that
culture-expanded cells from patients diagnosed for OA have the capacity to proliferate
and produce matrix proteins in the same quantity as ACT chondrocytes when placed in
a differentiation model.

Materials and methods

Cartilage harvest

Cartilage biopsies were harvested with a curved chisel from macroscopically affected
and unaffected surplus cartilage from seven patients with OA (age 64 to 83 years),
with OA grades 3 to 5 on the Ahlbäck scale [16], undergoing total knee replacement. The affected side was considered to be the femoral
condyle on the concave side of the knee deformity; that is, the medial condyle in
varus deformity and the lateral in valgus knees. In all patients the hip–knee–ankle
angle was determined from standing whole-leg radiographs (an angle of more than 180°
indicates a valgus knee deformity). The harvested biopsies were transported to the
cell culture laboratory in sterile saline solution (0.9% NaCl; Fresenius Kabi, Uppsala,
Sweden) supplemented with gentamicin sulphate (50 mg/l; Gibco, Paisley, Renfrewshire,
UK) and amphotericin B (250 μg/ml; Gibco). Part of the cartilage biopsy was processed
for histology, blinded and scored by two independent experienced researchers in accordance
with a modified (biopsies without subchondral bone) Mankin scale [17], with a maximum score of 13. The rest of the biopsy was used for cell culture as
described below. The donation of surplus cartilage was approved by the ethical committee
at the Medical Faculty at Gothenburg University.

Cell culture

The chondrocytes were isolated from the surrounding matrix by mechanical mincing of
the tissue with scalpel followed by enzymatic treatment overnight with collagenase
(0.8 mg/ml; Worthington Biochemical Corp, Lakewood, NJ, USA) in Ham's F-12 medium
(Invitrogen, Lidingö, Sweden), at 37°C in 7% CO2/93% air. The isolated cells were seeded at 104 cells/cm2 in culture flasks (Costar; Corning Incorporated, Corning, NY, USA) in DMEM/F12 medium
(Invitrogen) supplemented with L-ascorbic acid (0.025 mg/ml; Apotekets produktionsenhet,
Umeå, Sweden), gentamicin sulphate (50 mg/l; Gibco), amphotericin B (250 μg/ml) and
L-glutamine (2 mM; Gibco) with the addition of 10% human serum [18]. In brief, the human serum was collected in transfusion bags (dry pack; JMS, Singapore)
from healthy blood donors. The serum was left to coagulate overnight at 4 to 8°C,
centrifuged, sterile filtered, divided into aliquots and frozen until use. The first
medium change was made on day 6 and thereafter twice a week. When the cells reached
80% confluence, they were subcultured and frozen. Thawed cells were subcultured into
new flasks (Costar) at a density of 4 × 103 cells/cm2.

Three-dimensional pellet culture

After passage 1, the cells were cultured in a 3D pellet culture system as described
previously [15,19]. On days 7 and 14, the pellets were fixed in Histofix™ (Histolab Products AB, Göteborg,
Sweden), dehydrated and embedded in paraffin. Sections 5 μm thick were cut and placed
on microscope slides (Superfrost Plus; Menzel-Gläser, Braunschweig, Germany), deparaffinized
and stained with Alcian blue/van Gieson or immunohistochemically with anti-type I
collagen and anti-type II collagen antibodies.

Immunohistochemistry of pellets

Deparaffinized sections were digested with hyaluronidase, 8,000 units/ml (Sigma, St
Louis, MO) in 0.1 M PBS for 60 min at 37°C and blocked with 3% BSA (Sigma) in PBS
for 5 min. The primary antibodies (anti-type I and II collagen; ICN Biomedicals, Aurora,
OH, USA), diluted 1:150 in PBS containing 3% BSA, were incubated with the sections
for 1 hour at room temperature (20–22°C). The secondary antibody, peroxidase-conjugated
goat anti-mouse (1:150; Jackson Immunoresearch Laboratories, West Grove, PA, USA)
were applied to the sections for 1 hour at room temperature. A substrate kit (Vector
VIP; Vector Laboratories, Burlingame, CA, USA) was used for visualization and the
results were analysed with a Nikon Optiphot2-pol microscope (Nikon Instruments Inc,
Melville, NY, USA). Goat cartilage and bone explants were used as a positive control;
for a negative control the primary antibodies were omitted.

Biochemical analysis of pellets

On days 7 and 14, pellets were digested in papain (Sigma) solution (0.3 mg/ml in 20
mM sodium phosphate buffer, pH 7.4, containing 1 mM EDTA and 2 mM dithiothreitol)
for 60 min at 60°C. The digested pellets were then mechanically dissolved by vortex-mixing
and further analysed for DNA, glycosaminoglycan and hydroxyproline content as described
previously [15]. All biochemical analyses was performed on triplicate pellets.

Isolation of total RNA

Total RNA was isolated from cells cultured in a monolayer (passage 1) and from day
7 pellets with the use of an RNeasy mini kit (Qiagen, Hilden, Germany) in accordance
with the manufacturer's description. Before RNA isolation, the pellets were collected
in a 1.5 ml micro-tube (Sahrstedt, Nümbrecht, Germany) containing RLT buffer (Qiagen)
and disrupted by sonication. To remove cell debris and cartilage matrix proteins a
QIAshredder column (Qiagen) was used. Contaminating genomic DNA was removed from the
isolated RNA by using a DNA-free kit (Ambion, Huntingdon, UK) and total RNA content
and purity were determined spectrophotometrically at 260 and 280 nm. In general, A260/A280 ratios of about 2 were considered to indicate acceptable purity of the samples [20].

Reverse transcription in vitro was performed with 500 ng of total RNA with the use of random hexamer primers and
TaqMan Reverse Transcription reagents (Applied Biosystems). Real-time PCR was performed
with 5 μl of diluted (1:10) cDNA corresponding to 10 ng of RNA, 15 μl of TaqMan Universal
PCR master mixture (Applied Biosystems), 1× assay-on-demand mixes of primers and TaqMan
MGB probes. All samples were analysed in triplicate and PCR was performed in optical
96-well microtitre plates (Applied Biosystems). After an initial denaturation step
at 95°C for 10 min, the cDNA products were amplified with 40 PCR cycles consisting
of a denaturation step at 95°C for 15 s and an extension step at 60°C for 1 min.

To analyse the real-time PCR data, a standard curve method was used. The data were
analysed with ABI Prism 7000 SDS software (Applied Biosystems). For each sample, the
Ctsample values were determined as the cycle number at which all samples were in the exponential
phase of amplification. By using the formulas below, a value (Y) was obtained as a measure of the gene expression correlated to the standard curve
for that particular gene: X = (Ctsample - Intercept value)/Slope value; X10 = Y. The Y value for each cDNA sample and target sequence was divided by the Y value from the housekeeping gene (18S) for that particular sample to derive a ΔCt value (PE-ABI; Sequence Detector User Bulletin 2).

Statistical analysis

Biochemical differences between donors and chondrocytes isolated from affected and
unaffected were analysed with a two-sided Student's t-test (two-sample equal variance). P < 0.05 was considered significant. All analyses were performed with cell samples from
at least three separate donors unless otherwise indicated; as a comparison, surplus
cells from three or four donors undergoing ACT were used [15].

Results

After histological preparation, four of the seven isolated biopsies were evaluated
on the Mankin scale for severity of OA [17]. The score in these samples varied from 1.5 to 11, and in two of the patients a significant
difference was found between the affected pathological and unaffected non-pathological
side of the joint (Fig. 1). After mechanical and enzymatic isolation of the chondrocytes from the biopsies,
no difference could be observed in the average number of cells per milligram of cartilage
between the affected and unaffected sides (Table 1). These numbers did not differ from the average number of cells isolated from ACT
patients [21].

Figure 1. Histology of biopsies. The figure shows sections stained with Alcian blue/van Gieson.
The biopsies originate from two representative autologous chondrocyte transplantation
patients (ACT) (a, b) and from the unaffected and affected side from two patients with osteoarthritis (OA)
undergoing total knee replacement (c–f). (c, e) Biopsy from one OA donor (female, aged 81 years) with a Mankin score of 1.5 on both
sides. (d, f) Biopsy from another OA donor (male, aged 74 years) with Mankin scores of 5 (d) and
11 (f).

In the primary cultures of the isolated chondrocytes from the unaffected and affected
sides, floating matrix fragments were initially found in the affected cultures. These
fragments did not seem to affect the proliferation ability and disappeared after the
first change of medium. The cells from the unaffected and affected sides expanded
with, on average, 0.21 and 0.22 cell doublings per day, respectively. After one passage
(4.3 cell doublings) and 3 weeks of culture, 106 primary cells isolated from a 400 mg biopsy were expanded into 20 million cells.

When the expanded cells were cultured in serum-free medium in a redifferentiation
model they formed spherical pellets overnight. During this shift from two-dimensional
culture to 3D culture, the cells expressed increasing amounts of type II and type
X collagen, whereas the expression of type I collagen was unchanged or slightly decreased
(Fig. 2). No difference in expression of these typical cartilage genes was observed between
affected, unaffected and ACT donors.

The shift from a proliferative to a matrix-synthesizing state was also demonstrated
by an increase in the size of pellets from day 1 to day 14. The histological sections
of these pellets showed flattened cells on the surface and round cells in the centre
(Fig. 3). Spindle-shaped cells were found in the central part of the pellet in some donors,
and the frequency of spindle-shaped cells was greater in samples isolated from biopsies
with high Mankin scores. In the pellets, sulphated proteoglycans were detected by
Alcian blue/van Gieson staining at both days 7 and 14 in all donors (Fig. 3). Metachromatic staining was normally found throughout the whole pellets, but slightly
weaker staining was found in the day 7 pellet from the sample with the highest Mankin
score (data not shown).

The increase in pellet size during the culture period was accompanied by a significant
increase in DNA amounts in all samples, except from one donor with Mankin score 11
on the affected side, between days 7 and 14 (data not shown). During the same period
there was an increase in proteoglycan accumulation in each cell in 63% of all samples.
At day 14 no difference could be observed in the amount of proteoglycans per cell
in pellets from the unaffected and affected sides (P > 0.05). In a comparison between OA chondrocytes and those from patients undergoing
ACT only one sample, from the affected side of a female aged 81 years, had a significantly
lower content of proteoglycans (P < 0.05; Fig. 4a).

Figure 4. Biochemical analysis of pellets. Results are shown of the measurement of cartilage
matrix protein accumulation in day 14 pellets normalized to cell number (DNA) in cells
from the unaffected (filled bars) and affected (open bars) sides of four consecutive
donors; the sex and age (in years) of the donors are indicated. Glycosaminoglycan
(GAG) (a) and hydroxyproline (as a measure of total collagen content) (b) are shown as amounts per microgram of DNA. Results are means ± SD for three identical
pellets. The amounts are compared with the mean value for four sequential autologous
chondrocyte transplantation patients (ACT). An asterisk indicates a significant difference
(P < 0.05) between ACT and osteoarthritis chondrocytes.

The ability of the culture-expanded cells to form collagens in the pellet model was
analysed biochemically and by immunoreactivity to type I and type II collagen (Fig.
3). The total amounts of collagen per cell were significantly lower in all OA samples
than in those from ACT patients (Fig. 4b). By immunohistochemistry, type II collagen was detected in all donors at day 14,
on both affected and unaffected sides, without any correlation with Mankin score.
In the immunohistochemical analysis for type I collagen, both samples from one donor
(a male aged 74 years) with Mankin scores of 6 (unaffected) and 11 (affected), stained
positive at days 7 and 14. The other samples were only weakly positive at day 14.

To test the potential of using Hyaff-11 as a scaffold for the delivery of chondrocytes,
the scaffold was seeded from the top with two different concentrations of cell suspensions
of OA samples and samples from ACT patients. After the use of this technique, the
chondrocytes could be detected throughout the whole thickness of the scaffold, but
higher concentrations of cells were observed on the side of the scaffold from which
the cells had been seeded (Fig. 5). This cell distribution was more obvious in the scaffolds seeded with OA chondrocytes
than in those seeded with ACT chondrocytes.

Figure 5. Histology of cell-seeded scaffolds. The figure shows scaffolds seeded with chondrocytes
from one representative autologous chondrocyte transplantation (ACT) donor (a–c) and one osteoarthritis (OA) donor (unaffected) at two different cell seeding densities,
106 cells/cm2 (d–f) and 5 × 106 cells/cm2 (g–i). Accumulation of proteoglycans is shown with Alcian blue/van Gieson stain and the
presence of type II collagen is indicated by red staining within the scaffold. Panels
(b), (e) and (h) are higher magnifications of portions of (a), (d) and (g), respectively.

Attached to the hyaluronic acid, the chondrocytes redifferentiated within the scaffold,
as seen by the secretion of proteoglycans and the synthesis of type II collagen (Fig.
5). The expression of cartilage proteins was more obvious on the surface of the scaffolds
seeded with the high cell density than those seeded with the low cell density, as
shown by the increased intensity in staining with Alcian blue/van Gieson and in staining
for type II collagen (Fig. 5).

Discussion

Chondrocytes isolated from OA cartilage are able to proliferate in a monolayer and
redifferentiate in 3D models, demonstrating properties similar to those of non-OA
chondrocytes used for ACT. This indicates that culture-expanded autologous chondrocytes
from OA patients could potentially be used for resurfacing articular cartilage.

In this paper we studied the potential of chondrocytes isolated from patients with
developed OA. During the initial monolayer culture, chondrocytes are extracted from
their normal 3D environment and exposed to an artificial environment consisting of
a plastic surface, culture medium and serum. The plastic provides a substrate for
the growth of the anchorage-dependent cells and the culture medium stabilizes pH and
osmolarity and supplements the cells with trace compounds and energy sources (pyruvate
and glucose). The added serum contains high levels of growth factors released by cells
and platelets during the coagulation process of whole blood and has the ability to
stimulate cell proliferation [18]. In this artificial environment enriched in growth factors the chondrocytes proliferate,
dedifferentiate and lose their phenotype. The ability of these dedifferentiated cells
to redifferentiate into the chondrogenic phenotype has been proven to be affected
by the growth factors used during expansion [22] and the number of cell divisions [23].

In ACT treatments, 106 cells/cm2 are implanted into the defects under a covering of periosteum or type I/III collagen
membrane [24]. If this treatment were to be used for OA patients, most probably both the femoral
condyle and the tibial plateau would need restoration. This would mean that surfaces
about at least 25 cm2 in size should be treated. In the present study we were able to obtain, from a 400
mg cartilage biopsy taken from OA patients, 20 million cells within 3 weeks of culture.
This means that without exceeding the number of cell divisions, which could possibly
hamper the redifferentiation potential [25], it would be necessary to harvest about 500 mg of cartilage, which correlates to
a circular biopsy 7.2 mm in diameter on the basis of calculations of normal hyaline
cartilage [26]. The data in this study indicate that the biopsy could be harvested either from a
non-weight-bearing area or from the actual affected area during a cleanout pre-arthroscopic
procedure. However, 106 cells/cm2 greatly exceeds the cell density in adult cartilage, and the number of cells actually
needed for a successful scaffold-assisted cartilage repair has not been defined.

It has previously been demonstrated in several studies that cells isolated from OA
cartilage have limited proliferation capacity [27] and malfunctioning proteoglycan synthesis [10,11]. It was therefore a great surprise to us that we observed a proliferation rate similar
to that in samples from patients treated with ACT and no difference in the proteoglycan
secretion in chondrocytes isolated from affected and unaffected areas. All samples
had the further ability to produce type II collagen in the pellet model. Possible
explanations for this are that during the proliferation phase the cells are exposed
to an environment and to growth factors, which 'revitalizes' the cells [10], or simply that there is a positive selection of potent cells during the monolayer
culture.

Although the chondrocytes from OA patients analysed in this study produced a cartilage-specific
matrix, the ability of the chondrocytes to redifferentiate seemed be different from
that of chondrocytes isolated from ACT donors [15]. Whereas ACT chondrocytes, once placed in the 3D serum-free pellet model, stopped
their DNA synthesis and started to differentiate, the OA chondrocytes continued to
proliferate up to day 14. The proliferation was accompanied with significantly less
collagen production in all OA chondrocytes than in ACT chondrocytes (Fig. 4b). A shift from a differentiated phenotype to a proliferative state has further been
suggested as an explanation for the development of OA [9,28] and could possibly be reflected in the inability to redifferentiate seen in the pellet
model.

Another important issue in cell-based cartilage repair, especially for large defects,
is the positioning of cells in large defects. This can be done by delivering the cells
to the patient within a vehicle or a scaffold. Within the scaffold, which is preferably
biodegradable and has a controlled degradation time, the cells are able to attach
and to start producing cartilage matrix. In our study we observed that, within the
hyaluronic acid scaffold after 2 weeks of culture in serum-free medium, the OA chondrocytes
formed cartilage matrix proteins. This result concurs with previous studies with human
epiphyseal chondrocytes and chick embryonic sternal chondrocytes, in which an increased
expression of cartilage typical genes was observed in cultures with Hyaff-11 (scaffold
based on hyaluronic acid) [29].

The redifferentiation of the dedifferentiated cells was typically more obvious in
the scaffolds seeded with the high density of cells (25 × 106 cells/cm3), indicating that the cell density is important for the restoration of the chondrogenic
phenotype. The cell density and redifferentiation could also be important for matrix
production, because in the scaffolds seeded with a low cell density (5 × 106 cells/cm3) we observed a threefold to fivefold lower secretion of proteoglycans compared with
the pellet cultures (data not shown). Similar observations have been presented by
Puelacher and colleagues [30], who showed that at least 20 × 106 cells/cm3 were needed for good matrix formation within the scaffold.

Further, it is of great importance that the scaffold, when implanted into the joint,
has the ability to integrate with the surrounding cartilage and with the subchondral
bone. Integration with the subchondral bone could possibly be increased by the induction
of subchondral bleeding, for example by microfracture. However, the importance of
an uninjured subchondral bone plate for the integrity of the articular cartilage and
the ability to withstand mineralization has not been clarified.

The integration could also be altered by the grade of differentiation of the scaffold,
as demonstrated in a study made by Obradovic and colleagues [31]. They showed that the integration of tissue-engineered cartilage to articular cartilage
explants was better with immature (redifferentiated for 5 days) than mature (redifferentiated
for 5 weeks) cartilaginous explants. The positive immunostaining of type II collagen
seen in our scaffold seeded with the higher density of cells could indicate that the
cells had redifferentiated too far and that the implant would therefore be less integrative.
In contrast, in the treatment of large injuries, the scaffold needs to be able to
withstand mechanical load and shear forces from the time of implantation. These forces
can possibly be lowered by alignment of the mechanical axis (tibia osteotomy) to reduce
the weight bearing of the implant, but the scaffold will in any case be subjected
to mechanical stress and will have to be able to withstand this. A possible way of
strengthening the scaffold without redifferentiation would be to distribute the chondrocytes
more uniformly in the scaffold by improving the seeding method. Both spinner flask
and perfusion culture techniques have been shown to be superior to static cultures
[32].

Another reason that the implant has to withstand mechanical stress is that systematic
redifferentiation signalling, as part of the disease condition, could be impaired
within the OA joint. Redifferentiation and proteoglycan synthesis could instead be
stimulated by dynamic mechanical compression of the implant. The mechanical load could
possibly be gradually increased to adapt to the differentiation state of the implant
through specifically developed physiotherapy programmes, which will therefore probably
have an important role in the development of biological implants for OA.

Conclusion

We demonstrate in this paper that OA chondrocytes have the ability to proliferate,
redifferentiate and secrete cartilage-specific matrix proteins. We also show that
OA chondrocytes have an inability to shift definitely from a proliferative to a differentiating
state. How to change the cells from a proliferative to a collagen-secreting phenotype
needs to be explored further, especially when considering the importance of collagen
in maintaining the cartilage structure.

We further showed that the OA chondrocytes are able to bind to a scaffold, but further
studies will be needed to establish how far the cartilage in this scaffold should
be differentiated to be able to integrate with the surrounding cartilage and subchondral
bone and to withstand the mechanical forces applied within the joint.

The results in this paper give hopes for finding a cell-based autologous biological
treatment for young active patients with OA, but we have to remember that there is
no normal cartilage in OA and more research must be done before such a treatment can
be put into clinical practice.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

TT conceived of the study, coordinated the experiments, performed immunohistochemical
staining, performed the statistical analysis and drafted the manuscript. C Bengtsson
performed the cell culture and RNA preparations. C Brantsing performed immunohistochemical
stainings and the quantitative PCR analysis. ESJ participated in the design of the
study and gave clinical cell culturing input. LC isolated the biopsies and gave clinical
feedback. LP and MB provided critical clinical input to the study design and to the
manuscript. AL conceived of the study and gave critical comments on the manuscript.
All authors read and approved the final manuscript.

Acknowledgements

We acknowledge assistance from Mrs Helena Barreto in the biochemical analysis of the
pellets and scaffolds, and from Fidia Biopolymers in providing us with the Hyaff scaffold.